Lyme disease, the most common vector-borne disease in the Northern hemisphere, is transmitted to humans by ticks carrying the spirochete Borrelia burgdorferi. Enabled by a sudden influx of nutrients during tick feeding, the bacterium quickly remodels its surface to allow for efficient establishment of mammalian infection. The B. burgdorferi host-pathogen interface is dominated by lipoproteins that are peripherally anchored in the outer leaflet of the outer membrane via a lipid-modified cysteine. Recent studies in a mouse model of infection have shown that expression of outer surface lipoprotein C (OspC) is crucial for establishment of early infection. The overall objective of this study is to determine te structure function relationships of this important spirochetal virulence factor. OspC forms a homodimer on the surface of the cells, and its three-dimensional structure can be separated into three distinct domains: (i) a disordered N-terminal tether peptide, (ii) an alpha helical bundle scaffold, and (iii) a membrane-distal dome of helix-connecting loops. Yet, it remains unclear how these three domains are contributing to the lipoprotein's biological function in bacterial colonization and dissemination. OspC belongs to the same protein fold family as the relapsing fever Borrelia Vsp proteins, which are involved in immune evasion and tissue tropism. In fact, the crystal structure of Borrelia turicatae Vsp1 deviates from OspC only slightly but most significantly in the helix-connecting loops. We therefore hypothesize that OspC's dome residues are crucial for OspC's specific function during early infection. Furthermore, we hypothesize that the tether peptide, in addition to providing protein sorting information (see our published work), evolved to properly position the virulence factor within the bacterium's surface proteome through optimization of its length. We will test these hypotheses in the following two specific aims: 1. T determine the role of OspC dome residues in colonization and dissemination by testing if Vsp proteins or Vsp scaffold-OspC dome chimeras can functionally replace OspC both in binding of host molecules in vitro as well as in in a mouse model of infection. Ultimately, the minimal OspC dome domain required for function will be identified. 2. To determine the role of OspC lipoprotein tether length in virulence factor accessibility and function by testing existing and newly generated OspC tether length mutants for functions in vitro as well as in a competitive mouse infection assay. These studies will (i) achieve further milestones in our investigation of spirochetal surface lipoprotein structure- function, (ii) significantly increase our understanding f the early events during mammalian infection with Borrelia burgdorferi, and (iii) yield important clues that may ultimately translate into the design of novel intervention strategies for Lyme disease.